Suppressor of Cytokine Signaling 1 Regulates an Endogenous Inhibitor of a Mast Cell Protease

Subburaj Ilangumaran,Jason Raine,Dina Finan,Robert Rottapel

doi:10.1074/jbc.m308382200

Abstract

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of c-Kit and interleukin-3 (IL-3) receptor signaling. We examined the role of SOCS1 in regulating IL-3-induced cell growth of primary bone marrow-derived mast cells (BMMCs) from SOCS1-/- mice. Instead of showing increased proliferation, SOCS1-deficient BMMCs responded poorly to IL-3 and stem cell factor. SOCS1-/- BMMCs showed increased apoptosis and defective cell cycle entry. We show that the growth retardation of SOCS1-/- BMMCs was due to a cell intrinsic defect. Protein tyrosine phosphorylation following IL-3 stimulation was markedly diminished in SOCS1-/- BMMCs. Intriguingly, JAK2 and STAT5 proteins were selectively diminished in SOCS1-/- BMMCs, which also showed lower molecular mass products of p85 and Vav suggesting proteolytic degradation. Incubation of the SOCS1-/- BMMC lysate with STAT5, p85, and Vav immunoprecipitated from SOCS1+/+ cells directly demonstrated the dysregulated proteolytic activity in SOCS1-/- BMMCs. The proteolytic activity in SOCS1-/- BMMCs was selectively inhibited by phenylmethylsulfonyl fluoride and soybean trypsin inhibitor, suggesting that the protease regulated by SOCS1 is a tryptase. The dysregulated tryptase in SOCS1-/- BMMCs is unlikely to be mMCP6 or mMCP7, because the enzyme activity was not inhibited by Polybrene but was inhibited by normal mouse plasma. SOCS1+/+ BMMC lysate inhibited the proteolytic activity present in SOCS1-/- BMMC lysate, indicating that SOCS1-/- BMMCs lack an endogenous protease inhibitor. These results show that SOCS1 is required for the expression and/or stability of an endogenous protease inhibitor, which protects mast cells from their own proteolytic enzymes.

Highlights

The generation, survival, proliferation, and functions of most hematopoietic cells critically depend on cytokines [1, 2]
We used bone marrow-derived mast cells (BMMCs) from SOCS1Ϫ/Ϫ mice to address whether Suppressor of cytokine signaling 1 (SOCS1) is a critical regulator of IL-3 signaling
SOCS1-deficient Mast Cells Exhibit Retarded Growth Due to Decreased Proliferative Response to IL-3 and Stem Cell Factor—We have previously shown that SOCS1 is rapidly induced in primary BMMCs following stimulation with IL-3 or stem cell factor (SCF), and forced expression of SOCS1 inhibits SCF-mediated cell proliferation without interfering with cell survival signals delivered via the c-Kit receptor [16]

Summary

Introduction

The generation, survival, proliferation, and functions of most hematopoietic cells critically depend on cytokines [1, 2]. These signals are promptly attenuated to prevent excessive signaling and uncontrolled cellular stimulation This is achieved by several mechanisms regulating the JAK kinase activity and the functioning of the STAT molecules. The regulatory function of SOCS molecules is mediated by the binding of the SH2 domain to the activation loop tyrosine of the JAK kinases [11,12,13,14,15,16]. After the binding of the SH2 domain to phosphotyrosine, the pre-SH2 region is believed to occlude the substrate binding pocket of the kinase In this manner, SOCS1 functions as a competitive inhibitor of JAK kinase substrates. We observed that SOCS1-deficient BMMCs showed a poor proliferative response to IL-3 and SCF, because SOCS1 deficiency dysregulated mast cell proteolytic enzymes leading to selective degradation of several signaling molecules

Methods

Results

Conclusion